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Abstract

Background

A bacterial strain previously isolated from pyrite mine drainage and named BAS-10
was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole
carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics.
In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having
a high rhamnose content and metal-binding properties, whose biotechnological applications
were proven as very relevant.

Results

Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed
that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun,
combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry
procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic
cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations
using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from
these differential studies peculiar levels of outer membrane proteins, key regulatory
factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar
biosynthesis or required for citrate fermentation and stress response during anaerobic
growth on Fe(III)-citrate were revealed. The protein differential regulation seems
to ensure efficient cell growth coupled with EPS production by adapting metabolic
and biochemical processes in order to face iron toxicity and to optimize energy production.

Conclusion

Differential proteomics provided insights on the molecular mechanisms necessary for
anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun,
also revealing genes that can be targeted for the rational design of high-yielding
EPS producer strains.

Background

Several species of enterobacteria use citrate as sole carbon and energy source. This
capability is firstly due to appropriate transporters for citrate up-take, such as
the citrate-specific proteins CitH and CitS [1,2] or like the tripartite tricarboxylate transporter (TTT) TctABC system able to transport
several tricarboxylic acids into the bacterial cell [3] or like the ferric citrate transport system (the product of the fecABCDE operon) that shuttles the Fe(III)-citrate complex into the cytoplasm [4-7].

During aerobiosis, intracellular citrate is catabolized throughout the TCA cycle.
Under anaerobic conditions, when TCA cycle is down-regulated, enterobacteria species,
like Klebsiella pneumoniae and Salmonella typhimurium, can grow on citrate by a Na(I)-dependent pathway, forming acetic acid and CO2 as final metabolites [1,8,9]. Genes specific for anaerobic citrate fermentation, such as those coding for regulators
(citAB), catabolic enzymes (citCDEFG and oadGAB) and citrate transporters (citS and citW), have been identified in these bacteria [1,2,8,10]. The presence of sodium is essential for citrate symport by CitS and for the activity
of oadGAB gene products that form the oxaloacetate decarboxylase complex. In fact, oxaloacetate
decarboxylase converts oxaloacetate into pyruvate and pumps sodium externally to synthesize
ATP [1,2,8,9].

Generally, iron is one of the major limiting nutrients [11] and citrate-fermenting enterobacteria do not usually thrive on high concentrations
of Fe(III)-citrate as sole carbon and energy source [12]. Indeed, there are habitats where the abundance of Fe(III) is so high, like in pyrite
mine drainages, which represents one of the major elements to make rust-red the acidic
waters. In this case, iron can represent an environmental hazard for life, especially
for its oxidative properties and for the presence of other metals which increase the
total toxicity of mine drainages and cause a significant reduction of microbial biodiversity
[13-15]. Only specialized species can survive in extreme habitats with high heavy metal concentrations
and carbon-depleted conditions and Enterobacteraceae are not expected to survive in such environments. Nevertheless, an enterobacterial
strain was isolated under an iron mat formed by waters leached from pyrite mine drainages
of Colline Metallifere, Tuscany, Italy [16]. This isolate, named BAS-10, was tentatively identified as K. oxytoca on the basis of partial (422 nt) 16S rDNA sequence and API Enterotube test [16]. Unique among Klebsiella strains, BAS-10 can ferment and proliferate on Fe(III)-citrate as sole carbon and
energy source, forming acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) [12]. Under these growth conditions, BAS-10 produces an EPS made of rhamnose (57.1%),
glucuronic acid (28.6%) and galactose (14.3%), which shows metal-binding properties
[17,18]. Although extracellular EPS have been reported over recent decades and their composition,
structure, biosynthesis and functional properties have been extensively studied, only
a few have been industrially developed [19].

Chelating and sugar compositional properties of BAS-10 EPS are of high interest for
potential biomedical, food, and environmental applications [18-20]. Further studies on BAS-10 physiology may be useful to develop efficient fermentation
processes for EPS production. In order to investigate iron-dependent biochemical and
metabolic adaptations and regulatory networks thereof during anaerobic growth on Fe(III)-citrate
as the sole carbon source, a differential proteomic approach was used. In particular,
proteomic repertoires from BAS-10 grown on Fe(III)-citrate under anaerobiosis, on
Na-citrate under anaerobiosis and on Fe(III)-citrate under aerobiosis were comparatively
evaluated.

Results and discussion

Phylogenetic identification

Physiological studies [12], EPS composition and metal-binding activity thereof [17,18] revealed characteristic peculiarities of BAS-10 strain. Thus, a sequence of 1447
nt gene was generated from BAS-10 16S rDNA (Additional File 1) to perform phylogenetic clustering. Two ClustalW analyses were performed by using
the first twenty hits from Blast analysis, selecting only cultivable reference or
whole strains from NCBI database (http://blast.ncbi.nlm.nih.gov/webcite) [21], respectively. Phylogenetic threes revealed that BAS-10 is related to K. oxytoca reference strain (Figure 1) and can be clustered into a group comprising other five very close related strains
classified as K. oxytoca (Additional file 1 Figure S1). This investigation definitively confirmed previous taxonomical observations
on BAS-10 [16].

Figure 1.Phylogenetic analysis of BAS-10 strain performed by using 16S rDNA sequences. The first twenty hits from BLAST analysis performed by selecting only cultivable
reference strains were used to generate the phylogenetic three. The 16S rDNA sequence
of Streptomyces tendae was used as outgroup. Distance unit is based on sequence identity. NCBI accession
number of each 16S rDNA is reported after hyphen.

Proteins required for Fe(III)-citrate fermentation

The differential proteomic analysis revealed up-regulation of citrate lyase α- and
β-subunit (CitE and CitF), oxaloacetate decarboxylase α-subunit (OadA), pyruvate formate
lyase (PFL), phosphotrans-acetylase (PAT) and acetate/propionate kinase (ACK) in anaerobic
FEC with respect to both aerobic FEC and anaerobic NAC (Table 1; Figures 2 and 3). These proteins are known to be involved in anaerobic citrate fermentation, eventually
converting citrate to acetate with production of ATP. In particular, CitF and CitE
are part of the citrate lyase complex, the key enzyme in initiating the anaerobic
utilization of citrate, which is responsible for catalyzing the conversion of citrate
into acetate and oxaloacetate. The oxaloacetate decarboxylase complex, constituted
by alpha, beta and gamma subunits, catalyzes the second step of citrate fermentation
by converting oxaloacetate into pyruvate, the latter being the substrate of PFL, a
typical enzyme of enterobacteria growing under anaerobic conditions [25], that generates formate and acetyl-CoA. Phosphotrans-acetylase (PAT) catalyzes conversion
of acetyl-CoA into acetyl-P, which finally transfers the phosphate group to ADP to
yield acetic acid and ATP in the ACK-catalyzed last step of citrate fermentation.
Different forms of CitE, CitF, OadA and PFL were identified in K. oxytoca BAS-10 2D-protein maps (Table 1, Figures 2 and 3; Additional file 1 Table S1 and Figure S2). The occurrence of several protein forms having Mw and/or
pI different from the predicted one would imply that protein activity could be controlled
by post-translational modifications (PTMs), like covalently-bound charged small molecules
(as in the case horizontal spot trains) or proteolytic digestion (as in the case of
protein isoform with reduced Mw). Indeed, PTMs, like acetylation, succinylation and
radical formation have already been shown for PFL activity regulation [26-28], thus giving count for the generation of different protein spots in 2D-maps. In the
case of OadA, biotinylation in lysine, reported to be crucial for oxaloacetate decarboxylase
activity [29], could explain the slight increase of the measured Mw while the generation of reduced
Mw species is likely to be due to proteolytic fragmentation. Concerning CitE and CitF,
this is the first study suggesting PTM regulatory events. The differential analysis
showed that anaerobic conditions promote citrate fermentation enzymes accumulation,
similarly to what already reported in other enterobacteria [10,30,31]. In addition and more interestingly, during BAS-10 anaerobic growth, Fe(III) positively
controls the accumulation of citrate fermentative enzymes better than Na(I). In fact,
in K. oxytoca BAS-10 citrate fermentation appears much more up-regulated in presence of Fe(III)
than Na(I), making this process unique for this microorganism among enterobacteria
which usually ferment citrate in a NA(I)-dependent manner [1,8-10].

Occurrence of Fe(III) during anaerobic growth also modulated the abundance of many
central carbon metabolism enzymes. In particular, pyruvate kinase (PK), glyceraldehyde
phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TIM) were up-regulated
whereas TCA cycle enzymes, such as dihydrolipoyllysine-residue succinyltransferase
(SucB), malate dehydrogenase (MDH) and fumarate hydratase (FH) were down-regulated
in anaerobic FEC with respect to both aerobic FEC and anaerobic NAC (Table 1, Figures 2 and 3). Interestingly, fumarate reductase flavoprotein subunit (FrdA) was up-regulated
during the anaerobic growth on FEC with respect to both NAC and aerobic FEC (Figure 4). FrdA is part of complex II homolog menaquinol:fumarate oxidoreductase, which oxidizes
menaquinol and transfers the electrons to fumarate during bacterial anaerobic respiration,
with fumarate as the terminal electron acceptor [32], thus counteracting TCA cycle down-regulation. Altogether these data suggest that
to efficiently divert the carbon flux towards acetate and ATP production citrate fermentation
enzymes are up-regulated during anaerobic growth on FEC, whereas TCA cycle enzymes
are repressed (Table 1 and Figure 4). In addition, these data indicated that the increased catabolism of citrate throughout
a fermentative pathway is coupled to the synthesis of metabolic precursors necessary
for anabolic processes like sugar biosynthesis. In particular, the TIM product glycerone-P
is a precursor involved in rhamnose biosynthesis (Figure 4). Since rhamnose is the major sugar of EPS [17], the observed TIM up-regulation can represent an interesting link between central
carbon metabolites and EPS synthesis in Klebsiella. As a consequence of the increased anaerobic citrate fermentation, the production
of acetic acid may also determine an increment of H+ gradient across cell membrane, which positively affects the activity of the ATP synthase
complex. In agreement with this view, ATP synthase subunit B was observed as up-regulated
under anaerobic conditions in FEC medium, in the respect of both NAC and aerobic FEC
(Table 1, Figures 2, 3 and 4).

Central carbon and nitrogen metabolism key regulators

The proteomic comparison showed down-regulation in anaerobic FEC of EIIAGlc, a component of bacterial phosphoenolpyruvate (PEP): carbohydrate phosphotransferase
system (PTS), which was revealed as two protein spots differing for Mw in 2D-maps
(Table 1, Figures 2 and 3; Additional file 1 Table S1 and Figure S2). The differential regulation of EIIAGlc may be related to the differential regulation of central carbon metabolism enzymes.
In fact, in both Gram-negative and Gram-positive bacteria PTS consists of several
factors interacting with different regulatory proteins thus controlling glucose metabolism
and many other cellular functions [33]. In particular, EIIAGlc is the central processing unit of carbon metabolism in enteric bacteria since it
is involved in the regulation of adenylate cyclase (AC) and therefore in carbon catabolite
repression trough the control of the catabolite repressor protein (CRP) (Figure 5). In addition, it also interacts with several non-PTS permeases and glycerol kinase
to inhibit their activity (inducer exclusion) [33].

Moreover, during anaerobic growth, the down regulation of EIIAGlc may be related to the observed up-regulation of ArcA in FEC with respect to NAC (Table 1; Figures 2 and 3). ArcA, a negative response regulator of genes in aerobic pathways [34,35], is a central regulator of carbon metabolism which, from one hand, negatively controls
the expression of pts and of TCA cycle genes [33,36] (Figure 5), and, from the other one, it positively regulates the expression of genes, like
citAB, pfl and ack, necessary for fermentative metabolic activities under anaerobic conditions [34,37] (Figure 5). In addition to its role of activator of fermentative genes and inhibitor of aerobic
catabolism genes ArcA appears to regulate a wide variety of processes like amino acid
and sugar transport and metabolism, cofactor and phospholipids biosynthesis, nucleic
acid metabolism [34,38].

Interestingly, the observed down-regulation of EIIAGlc in anaerobic FEC with respect to both NAC and aerobic FEC seemed coupled with a differential
regulation of important players of nitrogen and amino acid metabolism. In particular,
GlnK, identified as three different spots in K. oxytoca BAS-10 2D-maps (Table 1, Figure 2 and 3; Additional file 1 Table S1 and Figure S2), is part of nitrogen regulatory system (ntr) which facilitates the efficient assimilation of nitrogen from a variety of compounds
into glutamine and glutamate as reported in many bacteria like E. coli[39]. The activity of GlnK, which is encoded by an operon encoding the ammonia transporter
AmtB too, is controlled by uridylylaton state. In nitrogen proficiency, not-uridylylated
GlnK form is the most abundant and interacts with and inhibits AmtB [40]. In nitrogen limitation, uridylylated GlnK inhibits the phosphatase activity of the
sensor membrane protein GlnL, that forms a two-component system with the regulatory
protein GlnG. Phosphorylated GlnG positively affects the expression of many ntr genes, including GlnK [39,41] (Figure 5). The difference in pI of the GlnK forms here identified gives count for protein
uridylylation, which decreases the predicted pI value of native protein form. The
protein form with decreased Mw may be due to the possible removal of a N-terminal
amino acid stretch as reported in Streptomyces coelicolor[42]. Interestingly, the putative uridylylated GlnK was down-regulated in anaerobic FEC
with respect to both anaerobic NAC and aerobic FEC thus suggesting that nitrogen limitation
was sensed by BAS-10 growing in anaerobic FEC. The GlnK abundance was in agreement
with the down-regulation in anaerobic FEC of carbamoyl-phosphate synthase small subunit
(CPSase) in the respect of both NAC and aerobic FEC. In fact, CPSase, an enzymes involved
in pyrimidine and arginine biosynthesis, regulates nitrogen disposal by catalyzing
synthesis of carbamoyl phosphate from ammonia or glutamine and carbamate [43].

Carbon and nitrogen metabolism could also be associated to the regulation of membrane
proteins participating to amino acid up-taking, like the glutamine ABC transporter
periplasmic-binding protein (GlnH) and glutamate and aspartate transporter subunit
(DEBP), down-regulated in anaerobic FEC with respect to both NAC and aerobic FEC,
or arginine 3rd transport system periplasmic-binding protein (ArtJ) and leucine ABC transporter subunit
substrate-binding protein (LivK), up-regulated in anaerobic FEC with respect to both
NAC and aerobic FEC. In many bacteria, nitrogen and carbon metabolism are linked to
cell energetic state through the conversion of TCA cycle intermediate alpha-ketoglutarate
and the amino acid glutamate [44-46]. In K. oxytoca BAS-10 growing in anaerobic FEC, carbon metabolism regulators, like ArcA and CRP,
and nitrogen assimilation factors, such as GlnG, could be coordinately controlled
(Figure 5) in order to balance amino acid content and to promote biomass accumulation.

Redox balance and stress response proteins

Two isoforms of superoxide dismutase Mn-dependent (Mn-SOD) were down-regulated in
anaerobic FEC with the respect to both aerobic FEC and anaerobic NAC (Table 1, Figure 2 and 3; Additional file 1 Table S1 and Figure S2). Mn-SOD is devoted to destroy radicals which are normally
produced within the cells. Interestingly and in agreement with what already observed
in E. coli[47] and S. typhimurium[48], the presence of iron causes the accumulation of a Fe-SOD whose activity is devoted
to contrast iron dependent free radical formation. In particular, Fe-SOD had an abundance
level not depending on oxygen, since their relative amount was the same during aerobic
and anaerobic growth on FEC. Transcriptional regulation of both SOD genes is reported
to be mediated by the Ferric uptake regulator (Fur). Fur controls the transcription
of genes, like fecABCDE, involved in iron homeostasis and participates also in the transcriptional regulation
of many other genes, such as TCA cycle and PTS genes [49] (Figure 5). Indeed, Fur is mainly a transcriptional repressor when it binds Fe(II). Anyway,
by repressing the expression of the non-coding RNA Ryhb, that negatively controls
mRNA translation into proteins, consequently Fur may also indirectly exert a positive
gene regulatory effect as observed in E. coli[50]. This phenomenon could also occur in K. oxytoca BAS-10 since both fur and ryhb homologues were identified in Klebsiella genus (BLAST analysis, data not shown). Thus, it is likely that the Fe(III)-dependent
accumulation of Fe-SOD under anaerobiosis may depend on Fur action to counteract metal
toxicity, as observed in E. coli[51].

Furthermore, two proteins, the alkyl hydroperoxide reductase subunit C (AHPC) and
the thiol peroxidase (TPX), contrasting reactive oxygen species (ROS), were differentially
regulated in K. oxytoca BAS-10. In particular, AHPC was down-regulated in anaerobic FEC with respect to aerobic
FEC, thus revealing an oxygen dependent control, while TPX was down-regulated in anaerobic
FEC with respect to anaerobic NAC, thus revealing a Fe(III)-dependent regulation.

Concerning stress associate proteins, the hyperosmotically inducible periplasmic (OSMY)
was down-regulated in anaerobic FEC with respect to both aerobic FEC and anaerobic
NAC. This protein was found associated with osmotic stress response during E. coli[52]. Other stress associated proteins, like DNA protection during starvation protein
(DPS) and the universal stress protein F (USF), were down-regulated in anaerobic FEC
with the respect to aerobic FEC showing an oxygen dependent regulation. In DPS, a
ferritin-like protein, binds to the chromosome and protects DNA from oxidative damage
by sequestering intracellular Fe(III) and storing it in the form of Fe(III)-oxyhydroxide
mineral [53].

Proteome data revealed that outer membrane proteins are differentially regulated under
anaerobic condition in presence of FEC or NAC. In particular, three isoforms of major
outer membrane murein-associated lipoprotein (Lpp), differing in pI value, were up-regulated
in anaerobic FEC. In E. coli, Lpp is synthesized as a precursor protein (prolipoprotein) and processed by a signal
peptidase after modification with the addition of di-acyl-glycerol and a fatty acid
chain [54]. No modification altering pI has been reported up to day being this study the first
one suggesting the possibility of such similar PTMs in Lpp. This finding was coupled
with the down-regulation of outer membrane OmpA. Indeed, OmpA was revealed as four
down-regulated full size isoforms and one and three protein fragments up- and down-regulated
in anaerobic FEC, respectively (Table 1). In E. coli OmpA acts as a low permeability porin and it is present in E. coli protein 2D-maps as different protein spots suggesting PTM control [55]. Interestingly, both Lpp and OmpA are reported to interact with PAL that is part
of Tol-PAL complex, a membrane-spanning multiprotein system that has been reported
playing several functions in Gram-negative bacteria, including transport regulation,
cell envelope integrity and pathogenicity [56]. In addition, Lpp has been reported to interact with TonB protein, which serves to
couple the cytoplasmic membrane proton motive force to the active transport of iron-siderophore
complexes, including the Fec system [57]. The differential regulation of Lpp and OmpA suggests differential roles for these
two proteins in controlling iron omeostasis, membrane transport and/or preventing
possible cellular damage.

In conclusion, this proteomic analysis revealed an iron-dependent regulation for several
stress-related proteins; altogether, these observations suggest the occurrence of
different concomitant mechanisms to contrast and/or prevent BAS-10 cell damage during
aerobic or anaerobic growth.

Conclusion

At the best of our knowledge, for the first time this study describes at the proteome
level biochemical mechanisms allowing the physiological adaptation of the enterobacterium
K. oxytoca strain BAS-10 [12,16-18] to sustain anaerobic growth on Fe(III)-citrate as sole carbon and energy source.
K. oxytoca BAS-10, isolated from pyrite mine drainages of Colline Metallifere (Tuscany, Italy)
and sharing the same ecological niche of specialized bacteria like Acidithiobacillus ferrooxidans[58], has the peculiarity of thriving on high concentrations of Fe(III)-citrate. This
character distinguishes it from other clinically-isolated enterobacteria, belonging
to K. oxytoca, K. pneumonia, S. typhimurium and Neisseria gonorrhoeae species, which are able to uptake and ferment anaerobically citrate as sole carbon
and energy source in a Na(I)-dependent manner only [1,10,12,59]. When anaerobically grown on FEC or NAC, K. oxytoca BAS-10 biomass production yields and citrate consumptions were very similar [16]. Under this condition, about half of the initial Fe(III) was reduced to Fe(II) [12,16], the highest levels observed so far with respect to other fermentation processes
[60,61].

Differential proteome analyses, carried out using anaerobic FEC as pivotal condition,
revealed two sets of proteins whose abundance is O2- and Fe(III)-dependent, respectively. The cross checking of these proteome data revealed
proteins whose abundance is specifically associated to anaerobic growth on Fe(III).
Interestingly, these data portrayed a coordinated regulation of citrate catabolic
enzymes during anaerobic growth on FEC. Citrate may enter the cell throughout several
kind of permeases, including Cit transporters [1,10], TctABC [3] and FecABCDE systems [4-7]. Then, it is catabolised via the TCA cycle or the citrate fermentation pathway under
aerobic or anaerobic growth, respectively. Under anaerobic conditions, the first pathway
is repressed, while the second one is favoured. In addition, a fascinating speculation
is that one BAS-10 citrate fermentation enzyme, the oxalacetate decarboxylase, can
be adapted to act as both Fe(II)- and Na(I)-depending pump to excrete Fe(II) and Na(I).
This hypothesis has to be verified by functional studies on BAS-10 oadGAB genes. Furthermore, PK, GAPDH and TIM abundance levels suggested an increased synthesis
of metabolic precursors necessary for anabolic processes, such as those yielding rhamnose,
linking together central carbon metabolism and EPS biosynthesis in Klebsiella. Differential regulation of metabolic enzymes was associated with variable cellular
levels of carbon metabolism key regulators ArcA and PTS EIIAGlc, and important players of nitrogen and amino acid metabolism, like GlnK. In this
context, ArcA plays a central role in regulating anaerobic carbon metabolism by controlling
the expression of citrate fermentation and TCA cycle enzymes directly or indirectly
– i.e. throughout PTS gene regulation – and nitrogen metabolism (Figure 5). Furthermore, during anaerobic FEC growth, ArcA-dependent gene regulation may be
combined to iron effect on gene expression trough the activity of Fur (Figure 5). Proteome results also suggest that metabolic pathway modulation seems coupled with
regulation of redox balance, stress response and outer membrane protein content. The
occurrence of various proteins involved in these processes is finely balanced to face
iron and pH toxicity, thus allowing K. oxytoca BAS-10 to survive in extreme habitats. Altogether, these data well correlate with
previous observations on other microorganisms able surviving in highly polluted environments
[62,63] or extremophile organisms living under extreme conditions [64,65], both generally activating multiple intracellular processes devoted to contrast environmental
toxic effects and optimize energy production.

According to the results presented here, K. oxytoca BAS-10 faces high iron concentration by various molecular adaptation mechanisms,
which are summarized in Figures 4 and 5. The coordinated regulation of metabolic pathways and cellular networks allows K. oxytoca BAS-10 to efficiently sustain bacterial growth by adapting metabolic and biochemical
processes in order to face iron toxicity and to ensure optimization of energy production.
In this context, EPS production is intrinsically related to bacterial biomass production
and metabolic and energy supply for anabolic reactions. Since K. oxytoca BAS-10 was proven to have a high potential for biotechnological applications [18-20], the data presented here could be used for redesigning fermentation strategies that
are difficult to be intuitively identified and approaching novel genetic targets to
be engineered for a rational design of high-yielding EPS producer strains.

Methods

For K. oxytoca BAS-10 cultures, two defined media were prepared containing the same mineral composition
(2.5 g NaHCO3, 1.5 g NH4Cl, 1.5 g MgSO4.7H2O, 0.6 g NaH2PO4, 0.1 g KCl, buffered at pH 7.6 with NaOH) and 50 mM Na-citrate (14.7 g.l-1), named NAC medium, or 50 mM ferric citrate (13.15 g.l-1), named FEC medium, as the sole carbon and energy source. BAS-10 was retrieved from
cryovials kept at −80°C in 25% glycerol in Nutrient Broth (NB) (Difco). One ml aliquots
of dense (abs600nm = 1.4) overnight NB pre-culture of BAS-10 were used to inoculate (1:100 v:v) FEC
or NAC medium cultivations. Aerobic cultivations were performed in flasks, aerated
by a magnetic stir bar (250 r.p.m.). Anaerobic cultivations were performed in pirex
bottles, previously fluxed with N2 and kept under anaerobiosis by sealed cap. Aerobic and anaerobic cultivations were
performed in parallel quadruplicates, at 30°C. Biomass samples for proteomic analysis
were harvested by centrifugation at late exponential growth stages. i.e. after 36
and 72 h of incubation in aerobic and anaerobic cultivations, respectively. For EPS
extraction and quantification, the procedures described by Baldi et al. (2009) [12] were followed.

Phylogenetic analysis

One microliter of diluted (1:10 in autoclaved distilled water) BAS-10 bacterial suspension
was used for PCR amplification of 16S rRNA gene by using fD1 and rD1 universal primers
[66] and 1.5 units of recombinant Taq DNA Polymerase (Invitrogen, Life Technologies),
according to manufacturer’s specifications. To perform bacterial cell lysis and DNA
denaturation, a treatment of 5 min, at 95 °C, was performed. Then, amplification steps
were carried out for 40 cycles consisting of 1 min at 95°C, 1 min at 55°C and 2 min
at 72 °C, with a final extension of 10 min at 72 °C. PCR fragments were purified by
using PCR clean-up kit (NucleoSpin, Macherey-Nagel) according to manufacturer’s specifications
and sequenced with the same primers at BMR Genomics (University of Padova, Italy).

BAS-10 16S rDNA sequence was compared using BLAST probing [67] of DNA sequences from the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHomewebcite) with default parameters, selecting either only cultivable reference strains (refseq_rna)
or whole database strains (rn/nt). ClustalX program [68] was used to align the homologous 16S rDNA gene sequences obtained from the database,
choosing the slow/accurate method for pairwise alignment. All positions containing
alignment gaps were eliminated in pairwise sequence comparisons by selecting the related
option. The 16S rDNA sequence of Streptomyces tendae was used as outgroup. Alienated sequences were then used to generate rooted trees
throughout Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm.

2D-DIGE analysis

Proteomes of K. oxytoca BAS-10 grown in FEC medium under anaerobiosis [−O2 and aerobiosis [+O2 or in NAC under [−O2 conditions were compared, using FEC medium under [−O2 as pivotal condition. To this aim, protein samples were labelled for 2D-DIGE analysis
using CyDyeTM DIGE minimal labelling kit (GE Healthcare, Sweden), as previously described [71]. Briefly, protein samples (40 μg) were labelled with 320 pmol of CyDye on ice in
the dark for 30 min. Labelling reaction was stopped by addition of 0.8 μl of 10 mM
lysine and incubation was continued on ice for 15 min., in the dark. Two samples out
of a total of four per each condition were labelled using Cy3 dye and the other two using Cy5 dye to account
for florescence bias. In addition, a standard protein pool was generated by combining
an equal amount of protein extracts from which six 40 μg protein aliquots were then
minimally labelled with 320 pmol Cy2 fluorescent dye (CyDyeTM DIGE, GE Healthcare), according to manufacturer’s instructions. Thus, a total of
six 2D-DIGE gels were performed containing a mix of Cy2-labeled pooled protein standard
and Cy3- and Cy5-labeled protein samples from cells incubated in FEC medium under
[−O2 and [+O2 and in NAC under [−O2 conditions, analysed in couples, i.e. FEC [−O2vs FEC [+O2, FEC [−O2vs NAC [−O2 and FEC [+O2vs NAC [−O2. For IEF, DeStreak rehydration solution (GE Healthcare), containing 0.68% v/v IPG
buffer (GE Healthcare) and 10 mM DTT (Sigma), was added to each mix up to a 340 μl
final volume. IEF was performed as previously described [57] using 4–7 pH range 18 cm-IPG strips (GE Healthcare) in an Ettan IPGphor III apparatus
(GE Healthcare). After IEF, IPG strips were incubated with an equilibration buffer
(6 M urea, 30% v/v glycerol, 2% w/v SDS, 0.05 M Tris–HCl, pH 6.8) containing 2% w/v
DTE for 10 min; thiol groups were then blocked by further incubation with equilibration
buffer containing 2.5% w/v iodoacetamide. Focused proteins were then separated by
using 12% SDS-PAGE, at 10°C, in a Hettan Dalt six (GE Healthcare), with a maximum
setting of 40 μA per gel and 110 V.

Protein visualization and image analysis

The 2D-gels were scanned with a DIGE imager (GE Healthcare) to detect cyanin-labeled
proteins, according to manufacturer’s instructions. Differential gel analysis was
performed automatically by using the Image Master 2D Platinum 7.0 DIGE software (GE
Healthcare), according to the manufacturer’s instructions. Protein spots were automatically
detected and then matched. Individual spot abundance was automatically calculated
from quadruplicated 2D-gels as mean spot volume, i.e. integration of optical density
over spot area, and normalized to the Cy2-labeled internal pooled protein standard.
Protein spots showing more than 1.5 fold change in spot volume (increased for up-regulation
or decreased for down-regulation), with a statistically significant ANOVA value (P ≤ 0.05), were considered differentially represented and further identified by MS
analysis.

Protein identification

Protein spots were excised from the 2D-gels, alkylated, digested with trypsin and
identified as previously reported [56]. Peptide mixtures were desalted by μZip-TipC18 (Millipore, MA) using 50% v/v acetonitrile/5%
v/v formic acid as eluent before MALDI-TOF-MS and nLC-ESI-LIT-MS/MS analysis.

In the case of MALDI-TOF-MS experiments, peptide mixtures were loaded on the MALDI
target, by using the dried droplet technique, α-cyano-4-hydroxycinnamic acid as matrix,
and analyzed using a Voyager-DE PRO mass spectrometer (Applied Biosystems, USA) operating
in positive ion reflectron, with an acceleration voltage of 20 kV, a N2 laser (337 nm) and a laser repetition rate of 4 Hz [72]. Final mass spectra, measured over a mass range of 700–4000 Da and obtained by averaging
400–800 laser shots, were elaborated using the DataExplorer 5.1 software (Applied
Biosystems) and manually inspected to get the corresponding peak lists. Internal mass
calibration was performed with peptides deriving from trypsin autoproteolysis. Tryptic
digests were eventually analyzed by nLC-ESI-LIT-MS/MS using a LTQ XL mass spectrometer
(Thermo, San Jose, CA) equipped with a Proxeon nanospray source connected to an Easy-nanoLC
(Proxeon, Odense, Denmark) [73,74]. Peptide mixtures were separated on an Easy C18 column (10–0.075 mm, 3 μm) (Proxeon).
Mobile phases were 0.1% v/v aqueous formic acid (solvent A) and 0.1% v/v formic acid
in acetonitrile (solvent B), running at total flow rate of 300 nL/min. Linear gradient
was initiated 20 min after sample loading; solvent B ramped from 5% to 35% over 15
min, from 35% to 95% over 2 min. Spectra were acquired in the range m/z 400–1800.
Acquisition was controlled by a data-dependent product ion scanning procedure over
the 3 most abundant ions, enabling dynamic exclusion (repeat count 2 and exclusion
duration 60 s); the mass isolation window and collision energy were set to m/z 3 and 35%, respectively.

MASCOT search engine version 2.2.06 (Matrix Science, UK) was used to identify protein
spots from an updated NCBI non-redundant database by using MALDI-TOF-MS and nLC-ESI-LIT-MS/MS
data and by selecting trypsin as proteolytic enzyme, a missed cleavages maximum value
of 2, Cys carbamidomethylation and Met oxidation as fixed and variable modification,
respectively. In the first case, a mass tolerance value of 40–80 ppm was selected;
in the second case, a mass tolerance value of 2 Da for precursor ion and 0.8 Da for
MS/MS fragments was chosen. MALDI-TOF-PMF candidates with a MASCOT score > 83 or nLC-ESI-LIT-MS/MS
candidates with more than 2 assigned peptides with an individual MASCOT score > 25,
both corresponding to P < 0.05 for a significant identification, were further evaluated by the comparison
with their calculated mass and pI values, using the experimental data obtained from
2D-DIGE.

Competing interests

The authors declare that they have no competing interests.

Authors’ contribution

GG carried out 16S rDNA amplification, phylogenetic analysis, 2D-DIGE analysis, gene
ontology and wrote the draft manuscript. FB designed and performed BAS10 cultivations
and helped to wrote the draft manuscript. GR carried out protein MS-identification.
MG helped to performe BAS10 cultivations. AC helped to perform 16S rDNA amplification,
phylogenetic analysis and 2D-DIGE experiments. AS supervised protein MS-identification
and revised the manuscript. AMP conceived and supervised the study and participated
in its design and coordination and revised the manuscript. All authors read and approved
the final manuscript.

Acknowledgements

This study was partially supported by Italian Ministery of Education, University and
Research (MIUR, ex 60%) to AMP and by funds from the “Rete di Spettrometria di Massa
della Regione Campania (RESMAC)” to AS. GG was supported by the European-funded project
“LAntibiotic Production: Technology, Optimization and improved Process” (LAPTOP),
KBBE-245066.

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